Determination Of 1-hydroxypyrene In Urine Research Articles

Application of a novel nylon needle filter-based solid-phase extraction device to determination of 1-hydroxypyrene in urine.

In this work, a simple and miniaturized solid-phase extraction device was constructed by connecting a commercial nylon needle filter to a syringe, which was applied for extracting 1-hydroxypyrene from a urine sample via hydrophobic and hydrogen bond interactions. The nylon membrane in the needle filter acted as the solid-phase extraction adsorbent, meanwhile, it filtered the particles in the urine sample. To obtain high extraction efficiency, key parameters influencing extraction recovery were investigated. The entire pretreatment process was accomplished within 5min under the optimal conditions. By coupling high-performance liquid chromatography-ultraviolet, a rapid, low-cost, and convenient nylon needle filter-based method was established for the analysis of 1-hydroxypyrene in a complex urine matrix. Within the linearity range of 0.2-1000 μg/L, the method exhibited a satisfactory correlation coefficient (R=0.9999). The limit of detection was 0.06 μg/L, and the recoveries from urine sample spiked with three concentrations (5, 20, and 100 μg/L) ranged from 105.8% to 113.1% with the relative standard deviations less than 6.7% (intra-day, n=6) and 8.9% (inter-day, n=4). Finally, the proposed method was successfully applied for detecting 1-hydroxypyrene in urine samples from college students, smokers, gas station workers, and chip factory workers. The detected concentration in actual urine samples ranged from 0.46 to 5.26 μg/L. Taken together, this simple and cost-effective nylon needle filter-based solid-phase extraction device showed an excellent application potential for pretreating hydrophobic analytes from aqueous samples.

Optimization of conditions for the sample preparation using mathematical planning for determination of 1-hydroxypyrene in urine using method of gas chromatography-mass spectrometry

Introduction. Biological monitoring of 1-hydroxypyrene is the most widely used method for evaluating the effects of PAHs on humans. The determination of 1-hydroxypyrene in urine is performed using gas chromatography-mass spectrometry (GC-MS). Material and methods. Sample preparation consists of extracting the analyte from the biological matrix by 2-fold liquid extraction with hexane, evaporation the extract to the dry residue in the nitrogen current, and re-dissolution of the dry residue in the silylating agent BSTFA. We used gas chromatograph Agilent 7890A with an HP-5MS capillary column and a mass-selective detector. 1-hydroxypyrene was identified as trimethyl silane on a mass chromatogram based on the retention time and intensity ratio of the registered ions. Results. Optimization of the conditions for liquid extraction of 1-hydroxypyrene was performed using mathematical planning, varying the mass of magnesium sulfate, the extraction time, and the extraction multiplicity. The planning matrix included eight experiments, and the degree extraction of 1-hydroxypyrene was used as an optimization parameter. Interpretation of the model showed the multiplicity of extraction to contribute more to the formation of the degree of extraction than the mass of magnesium sulfate and the extraction time. Discussion. The proposed method foк the sample preparation, based on the extraction of 1-hydroxypyrene by 2-fold liquid extraction with hexane for 2 min with the addition of 0.5 g of magnesium sulfate to the biological sample, allowed reaching the detection limit of 0.1 ng/ml. The evaluation of metrological characteristics showed RSD of the reproducibility to do not exceed 6.4%, the systematic error is not significant, and the accuracy index in the form of a total error is not higher than 15%. Levels of 1-hydroxypyrene in the urine of aluminum production workers are 53 - 414 times higher than in the control group. Conclusion. The choice of optimal conditions for liquid extraction of 1-hydroxypyrene by mathematical planning allowed developing a method for the determination this analyte in urine by GC-MS for biological monitoring.

Determination of 1-Hydroxypyrene as a Biomarker for the Effects of Polycyclic Aromatic Hydrocarbons in Urine by Chromatography–Mass Spectrometry

A simple and sensitive procedure has been developed for the determination of 1-hydroxypyrene in urine as a biological marker of the effects of polycyclic aromatic hydrocarbons. After enzymatic hydrolysis for conjugate cleavage, the analyte was extracted from the matrix by liquid–liquid extraction with n-hexane, the extract was evaporated to dryness, and the analyte was derivatized with the silylating reagent N,O-bis(trimethylsilyl)trifluoroacetamide into trimethylsilyl ether at room temperature. The trimethylsilyl extract was analyzed by capillary gas chromatography with mass-selective detection. Quantitative high-precision determination was achieved with the use of the isotope-labeled 1-hydroxypyrene-d9 reference standard. No matrix effects were observed. The detection limit was 0.02 ng/mL, and the linearity range was 0.1–100 ng/mL. Relative standard deviations were 0.044 and 0.064 under the conditions of repeatability and intralaboratory precision, respectively. The percentage recovery was 96–102%.

On-line flow injection molecularly imprinted solid phase extraction for the preconcentration and determination of 1-hydroxypyrene in urine samples

New analytical strategies tend to automation of sample pre-treatment and flow analysis techniques provided a number of enhanced analytical methods allowing high throughput. Flow techniques are usually faster, more robust and more flexible than their batch equivalents. In addition, flow methods use less sample and reagent amounts and reduce analytical costs and waste.A flow injection solid-phase extraction pre-concentration system using a molecularly imprinted polymer (MIP) packed micro-column was developed for the determination of 1-hydroxypyrene in human urine with fluorescence detection. The pre-concentration of 1-hydroxypyrene on the MIP was carried out based on the specific retention of analyte by on-line introducing the sample into the micro-column system. Methanol and dichloromethane mixture was used to elute the retained analyte for fluorometric analysis. Important influencing factors were studied in detail, in batch and in flow (MISPE procedure optimisation, sample and eluent volumes, flow rate, dimensions of MIP micro-column and amounts of packing material, etc).To the best of our knowledge, this is the first on-line flow injection molecularly imprinted solid phase extraction for the pre-concentration and determination of hydroxylate PAH metabolite in urine samples.The optimised method was successfully applied to the determination of 1-Hydroxypyrene in spiked urine samples, with recoveries in the range of 74–85% and RSD<4.6%. Under optimum experimental conditions, the linearity concentration range used was 10–400μgL−1, R2>0.996. We obtained limit of detection and quantification of 3.1μgL−1 and 10.5μgL−1, respectively.

Magnetic Solid Phase Extraction Followed by High Performance Liquid Chromatography for Determination of Urinary 1-Hydroxypyrene

Magnetic Solid Phase Extraction Followed by High Performance Liquid Chromatography for Determination of Urinary 1-Hydroxypyrene

AN IMPROVED METHOD FOR THE DETERMINATION OF URINARY 1-HYDROXYPYRENE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

An improved method based on solid-phase extraction and high-performance liquid chromatography with fluorescence detection was developed for the determination of urinary 1-hydroxypyrene (1-OHP). The solid-phase extraction procedure, the excitation and emission wavelengths for 1-OHP, and the pH of the mobile phase were optimized. Excitation and emission wavelengths of 239 and 392 nm, respectively, afforded much higher fluorescence response than the wavelengths reported in previous studies. Lowering the pH of the mobile phase significantly decreased the fluorescence response of 1-OHP but increased data reproducibility for urine samples. Under optimized conditions, the average relative standard deviation (n =5) of intra-day precision for 11 urine samples was 2.7% (range 0.8–4.0%). Because of the excellent data reproducibility, we expect the method to be useful for the determination of urinary 1-OHP, especially in biomonitoring projects involving high-volume screening.

Otimização e validação de uma metodologia analítica para determinação de 1-hidroxipireno em urina de cortadores de cana-de-açúcar

Este trabalho teve como objetivo otimizar e validar uma metodologia analítica para determinação de 1-hidróxipireno em urina de trabalhadores envolvidos na colheita da cana-de-açúcar. O método utilizado para determinação de 1-hidroxipireno em urina humana utilizado consiste na hidrólise enzimática, extração e clean-up por extração em fase sólida (SPE) e quantificação por cromatografia líquida de alta eficiência com detector de fluorescência (CLAE/Flu). Quatro tipos de cartuchos foram testados para verificação da porcentagem de recuperação. Urina de cortadores de cana-de-açúcar (não-fumantes e de ambos os sexos) foram coletadas no período da safra (n=39) e entressafra (n=34) da cana-de-açúcar. Os melhores resultados de recuperação foram atribuídos aos cartuchos C18. Os mesmos apresentaram recuperação entre 79% e 108%, com coeficiente de variação entre 5% e 10%. O limite de quantificação do método foi de 74 ng de 1-hidroxipireno por litro de urina. A metodologia otimizada e validada foi utilizada para determinação de amostras reais. Os resultados encontrados na urina dos trabalhadores no período da safra variaram de 0,026 a 2,3 μmol de 1-hidroxipireno por mol de creatinina. No período da entressafra os resultados variaram de 0,0023 a 0,38 μmol de 1-hidroxipireno por mol de creatinina.

Otimização e validação de uma metodologia analítica para determinação de 1-hidroxipireno em urina de cortadores de cana-de-açúcar

Este trabalho teve como objetivo otimizar e validar uma metodologia analítica para determinação de 1-hidróxipireno em urina de trabalhadores envolvidos na colheita da cana-de-açúcar. O método utilizado para determinação de 1-hidroxipireno em urina humana utilizado consiste na hidrólise enzimática, extração e clean-up por extração em fase sólida (SPE) e quantificação por cromatografia líquida de alta eficiência com detector de fluorescência (CLAE/Flu). Quatro tipos de cartuchos foram testados para verificação da porcentagem de recuperação. Urina de cortadores de cana-de-açúcar (não-fumantes e de ambos os sexos) foram coletadas no período da safra (n=39) e entressafra (n=34) da cana-de-açúcar. Os melhores resultados de recuperação foram atribuídos aos cartuchos C18. Os mesmos apresentaram recuperação entre 79% e 108%, com coeficiente de variação entre 5% e 10%. O limite de quantificação do método foi de 74 ng de 1-hidroxipireno por litro de urina. A metodologia otimizada e validada foi utilizada para determinação de amostras reais. Os resultados encontrados na urina dos trabalhadores no período da safra variaram de 0,026 a 2,3 μmol de 1-hidroxipireno por mol de creatinina. No período da entressafra os resultados variaram de 0,0023 a 0,38 μmol de 1-hidroxipireno por mol de creatinina. A metodologia validada mostrou-se adequada para determinação de 1-hidroxipireno em urina humana. Os dados obtidos permitem concluir que há forte correlação entre excreção de 1-hidroxipireno e os períodos de safra e entressafra da cana de açúcar.

Precision limits and interval estimation in the calibration of 1-hydroxypyrene in urine and hexachlorbenzene in water, applying the regression triplet procedure on chromatographic data

A method for the determination of 1-hydroxypyrene in urine and hexachlorbenzene in water applying the regression triplet in the calibration procedure of chromatographic data has been applied. The detection limit and quantification limit are currently calculated on the basis of the standard deviation of replicate analyses at a single concentration. However, since the standard deviation depends on concentration, these single-concentration techniques result in limits that are directly dependent on spiking concentration. A more rigorous approach requires first careful attention to the three components of the regression triplet (data, model, method), examining (1) the data quality of the proposed model, (2) the model quality and (3) the least-squares method to be used for fulfilment of all least-squares assumptions. For high-performance liquid chromatography determination of 1-hydroxypyrene in urine and gas chromatography analysis of hexachlorbenzene in water, this paper describes the effects of deviations from five basic assumptions The paper considers the correction of deviations: identifying influential points, namely, outliers, the calibration task depends on the regression model used, and the least-squares method is based on the assumptions of the normality of the errors, homoscedasticity and the independence of errors. Results show that the approach developed provides improved estimates of analytical limits and that the single-concentration approaches currently in wide use are seriously flawed.

Determination of Exposure to Bitumen and Fume from Bitumen in the Oil Industry Through Determination of Urinary 1-Hydroxypyrene

Since 1986, Shell routinely monitors occupational exposure of its operators to polycyclic aromatic hydrocarbons (PAHs) by determination of urinary 1-hydroxypyrene (1-HOPyr). Up till now, about 100 different studies in a wide variety of industrial settings, involving more than 4,000 samples in total, have been performed. The upper limit of the 95% confidence interval of background levels of 1-HOPyr in 121 workers without occupational exposure to PAHs was 0.51 μ mol/mol creatinine (median 0.11; range < 0.05 to 1.08 μ mol/mol creatinine). Due to the variation in composition of the PAH mixture (in particular the ratio between pyrene and carcinogenic PAHs) to which workers may be exposed in different processes ànd by different routes of exposure, it is not feasible to set a limit value for urinary 1-HOPyr. However, a value of 0.50 μ mol/mol creatinine can be used as an indicator whether or not occupational exposure to PAHs has occurred. This article focuses on a selection of studies in which operators were potentially exposed to to PAHs from bitumen and fume from bitumen. The selected studies involved manufacturing, maintenance of production facilities, and road tanker loading of bitumen. Studies over a number of years in a bitumen manufacturing plant indicated that exposures to PAHs as measured by 1-hydroxypyrene were not statistically significantly different from control values with median values of approximately 0.10 μ mol/mmol creatinine. However, occasionally values just over the the internal reference value (up to 0.72 μ mol/mol creatinine) were observed. Similar observations were made during road tanker loading and maintenance/cleaning operations although the value of 0.50 μ mol/mol creatinine was not exceeded in these studies. In several studies multiple pre- and post-shift samples were collected from individual operators during a number of consecutive days. Detailed analysis of the data strongly suggests that small changes over the shift, as seen during loading, manufacturing, and maintenance operations, were due to confounding factors (such as diet and smoking) rather than occupational exposure to PAHs. Several studies were performed to investigate the role of dermal exposure to PAH in more detail. In these studies industrial hygienists made detailed work observations to assess semi-quantitatively the extent of inhalatory and dermal exposure. In addition, studies in workers digging contaminated soil in which different forms of personal protective equipment providing variable degrees of respiratory and dermal protection during identical tasks, were used to show the effectiveness this equipment. Overal, these studies show that dermal exposure to PAHs may be significant in the total exposure to PAHs and that 1-HOPyr can be used to assess the efficacy of personal protective equipment. With appropriate personal protective equipment, exposure to PAHs, as assessed by the determination of urinary 1-HOPyr, could be reduced to background levels. The contribution of dermal exposure to the total uptake of PAHs, however, proved difficult to quantify exactly. In conclusion, both inhalatory and dermal exposure to PAHs from a variety of sources, including bitumen, can readily be assessed by the determination of 1-HOPyr in urine. Results from biomonitoring indicate that both inhalation and dermal exposure to PAHs during manufacturing and handling of bitumen is negligible.

Improved glucuronide hydrolysis in the determination of urinary 1-hydroxypyrene

The efficiency of the glucuronide hydrolysis in the determination of urinary 1-hydroxypyrene was investigated as a function of the reaction conditions. A significant improvement could be obtained by increasing the enzyme concentration described in the literature.

Estimation scheme for level-dependent uncertainty of analytical result: application for determination of 1-hydroxypyrene in urine

The estimation scheme of uncertainty of determination of 1-hydroxypyrene (1-OHP) in urine was developed analysing the main stages of the analytical procedure: (1) preparation of 1-OHP standards, (2) creation of the calibration curve for the high performance liquid chromatography (HPLC) analysis method with the evaluation of recovery, (3) measuring procedure of aliquot of urine, (4) adjusting the pH of aliquot and hydrolysis with enzyme, (5) solid phase extraction, (6) concentration of the extract, (7) injection of the extract to chromatograph and analysing by the HPLC method, (8) calculation of 1-OHP mass from the calibration curve, (9) calculation of 1-OHP concentration in urine. The evaluation of the uncertainty is based on quantification of individual components. Combined uncertainty was calculated using the law of propagation of uncertainties according to the EURACHEM/CITAC guidelines. Level dependence of the uncertainty arises from the calibration curve.

Determination of 1-hydroxypyrene in children urine using column-switching liquid chromatography and fluorescence detection

This study developed an acid hydrolysis method instead of using enzyme extraction, equipped with column-switching system for the pretreatment of samples, in the determination of 1-hydroxypyrene in the urine from children and pyrene in airborne particulates. We collected both types of samples from areas near a petrochemical industry and rural areas as reference. Samples were first treated with acid hydrolysis and followed by solvent extraction prior to being injected into the separation system for the determination with high performance liquid chromatography and fluorescence. A column-switching system was on-line with a C 18 separation column to remove matrix interference and obtain a stable baseline of the chromatogram. The eluent used to separate the 1-hydroxypyrene was 60% (v/v) aqueous acetonitrile solution. A fluorescence detector was used to monitor 1-hydroxypyrene at λ ex =348 nm and λ em =388 nm , and pyrene at λ ex =331 nm and λ em =390 nm . Both calibration graphs were linear with very good correlation coefficients ( r>0.999) and the detection limits were ca. 2 pg (5 ng/l). Results showed that there was a significant association between 1-hydroxypyrene levels in urine specimens and pyrene levels in airborne particulate samples ( r=0.68, P<0.05). The average levels of pyrene in the particulates (0.18 versus 0.09 ng/m 3) and of 1-hydroxypyrene in urine specimens (155.9 versus 110.2 ng/g creatinine) were higher for the petrochemical area than for the rural area. This method is stable and sensitive for measuring polycyclic aromatic hydrocarbons in environmental samples.

Derivative Matrix Isopotential Synchronous Fluorescence Spectroscopy for the Direct Determination of 1-Hydroxypyrene as a Urinary Biomarker of Exposure to Polycyclic Aromatic Hydrocarbons

Urinary 1-hydroxypyrene is a biomarker in the measurement of human exposure to polycyclic aromatic hydrocarbons. A rapid and simple derivative isopotential synchronous fluorescence method was developed for the direct determination of 1-hydroxypyrene in urine. A length of iso-intensity route was scanned on the three-dimensional fluorescence spectrum of urine and this result was combined with that from derivative technique. Thus the strong background signals of urine were removed and the 1-hydroxypyrene can be determined directly in urine without tedious pre-separation. The derivative isopotential synchronous fluorescence spectrum was directly obtained from a single scan on a spectrofluorometer, which further simplified isopotential synchronous fluorescence technique. The recoveries of 93% to 115% were obtained for 1-hydroxypyrene added to urine.

Methods for routine biological monitoring of carcinogenic PAH-mixtures

The ability of a biomarker to provide an assessment of the integrated individual dose following uptake through multiple routes is especially valuable for mixtures of polycyclic aromatic hydrocarbons (PAH), due to methodological and practical difficulties of collecting and analysing samples from the various environmental compartments like air, water and soil and various media such as diet, cigarette smoke and workroom air. Since 1980, a large variety of novel approaches and techniques have been suggested and tested, e.g. urinary thioethers, mutagenicity in urine, levels of PAH or PAH-metabolites in blood and urine and methods for determination of adducts in DNA and proteins. Two approaches are more frequently reported: PAH-DNA-adduct monitoring in blood cells and urinary 1-hydroxypyrene monitoring. A large research effort has been made to use the extent of binding of PAH to DNA as a biomarker of exposure. The 32P-post-labeling assay detects the total of aromatic DNA-adducts and the adduct level in white blood cells is claimed to be an indicator of the biological effect of the PAH-mixture. However, the levels of aromatic DNA-adducts may be subject to appreciable analytical and biological variation. The present technical complexity of the method makes it more convenient for research applications than for routine application in occupational health practice. Pyrene is a dominant compound in the PAH mixture and is mainly metabolised to the intermediary 1-hydroxypyrene to form 1-hydroxypyrene-glucuronide, which is excreted in urine. Since the introduction of the determination of 1-hydroxypyrene in urine as a biomarker for human exposure assessment in 1985, many reports from different countries from Europe, Asia and America confirmed the potential of this novel approach. The conclusion of the first international workshop on 1-hydroxypyrene in 1993 was that urinary 1-hydroxypyrene is a solid biological exposure indicator of PAH. Studies with a comparison of several biomarkers confirmed that 1-hydroxypyrene in urine is a valid and sensitive indicator of exposure. Periodical monitoring of 1-hydroxypyrene appears to be a powerful method in controlling occupational PAH-exposure in industries. The reference level and the biological exposure limit of 1-hydroxypyrene in urine are discussed.

Coupled-column high-performance liquid chromatographic method for the determination of 1-hydroxypyrene in urine of subjects exposed to polycyclic aromatic hydrocarbons

A coupled-column high-performance liquid chromatographic system for integrated, on-line sample processing and the determination of free and conjugated 1-hydroxypyrene in urine has been developed. The method is based on a “tailor-made” copper phthalocyanine-modified porous-glass precolumn packing material, which allows a direct and repeated injection of urine samples and a selective enrichment of trace amounts of particular components. The fully automated method has a low detection limit (0.01 pmol), a quantitative and matrix-independent recovery and a highly reliability, as shown by an interlaboratory comparison of methods.